Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.